Oral vitamin D3 supplementation enhances antiviral immunity in chickens challenged with fowl adenovirus serotype 4

Vitamin D is increasingly recognized as an important regulator of innate and adaptive immunity, yet its antiviral role in chickens during fowl adenovirus serotype 4 (FAdV-4) infection remains unclear. In this study, we investigated the effects of oral vitamin D 3 supplementation on humoral immunity, tissue viral load, and immune-related gene expression in FAdV-4-infected chickens. Ninety 8-day-old specific pathogen-free chickens were randomly divided into two groups (n = 45/group) and fed a basal diet. The experimental group received oral vitamin D 3 dissolved in 0.1 mL soybean oil at a dose equivalent to 3000 IU/kg diet daily for 3 weeks, whereas the control group received soybean oil alone. After supplementation, all birds were challenged with FAdV-4. Vitamin D 3 supplementation significantly reduced tissue viral loads and increased serum antibody levels compared with the control group. At the transcriptional level, vitamin D 3 modulated key innate and adaptive immune markers, including Toll-like receptors (TLRs), avian β-defensins, and cytokines. It downregulated several TLRs and reduced the pro-inflammatory cytokines interleukin (IL)-6 and IL-8, while increasing the antiviral cytokines interferon-γ and IL-1β. Vitamin D 3 also increased vitamin D receptor expression, decreased inducible nitric oxide synthase, and reduced the apoptosis regulators B-cell lymphoma-2 (BCL-2) and Bcl2-associated X protein in selected tissues. In conclusion, vitamin D 3 supplementation enhanced antiviral defense against FAdV-4 infection, possibly by promoting humoral immunity and modulating innate immune and apoptosis-related responses. These findings provide new insight into the role of vitamin D 3 in limiting FAdV-4 infection and support its potential as a nutritional strategy for controlling viral diseases in poultry.